POS0096 HYPOXIA AND MPO-ANCA IgG INDUCED BIOPHYSICAL CHANGES IN NEUTROPHILS MAY PROMOTE ENDOTHELIAL INJURY IN ANCA ASSOCIATED VASCULITIS
Authors: N. Pisacano, A. Dhutia, S. Rothery, S. P. Mcadoo, J. Guck, C. P. Pusey, E. Chilvers, A. Cowburn, K. Lodge, M. Prendecki
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> ANCA associated vasculitis (AAV) is characterised by neutrophil-mediated endothelial damage. Neutrophil deformability impacts their ability to traffic through microvasculature. Distinct biophysical properties of neutrophils in patients with active AAV, e.g. in response to ANCA or hypoxia in inflamed tissues, may contribute to endothelial injury. <h3>Objectives</h3> Investigate how MPO-ANCA IgG (ANCA) treatment and/or hypoxia affect the actin cytoskeleton or biophysical properties of neutrophils, and whether this phenotype is replicated in patients with active AAV. <h3>Methods</h3> Whole blood and isolated neutrophils from patients with active AAV (AAV), patients with AAV in remission (rAAV) or healthy controls (HC) was analysed using real time deformability cytometry (RT-DC), a novel microfluidic technique able to identify individual cell biophysical profiles. Deformation was calculated using ShapeOut. Neutrophils were isolated using immunomagnetic separation. Cells were primed with TNF (2 ng/mL, 15 min) and stimulated with healthy donor IgG (CIgG), or ANCA IgG (100 ug/mL, 1 h) under normoxia (21% O<sub>2</sub>) or hypoxia (5% O<sub>2</sub>). Cells were stained for F actin (AF488 Phalloidin) and imaged using confocal microscopy. Cell area and total percentage phalloidin staining was analysed (ImageJ). <h3>Results</h3> AAV neutrophils (n=27) were significantly stiffer than rAAV (n=25) or HC (n=15), displaying decreased deformation (median 0.085, 0.086, and 0.076 au for HC, rAAV, and AAV respectively, p=0.0001). There was an inverse correlation between AAV neutrophil deformation and disease activity as measured by BVAS score (r=-0.573, p=0.032). This phenotype of decreased deformation was replicated by ANCA stimulation of HC neutrophils (median 0.086, 0.085 and 0.082 for untreated, CIgG, and ANCA respectively, p=0.05). Hypoxia prevented the effect of ANCA stimulation on HC neutrophils (median 0.078, 0.077, and 0.079 au for untreated, CIgG, and ANCA respectively, ns). ANCA stimulation of HC neutrophils increased cell area (mean 51.56/63.07/88.61 um<sup>2</sup>, p=0.0008) and total actin signal (mean 27.61/25.86/44.30%, p=&lt;0.0001) (untreated, CIgG, ANCA respectively, Fig 1A-C). At baseline, hypoxic neutrophils were larger with increased actin staining. Under hypoxia the effect of ANCA stimulation was diminished, with a smaller increase in area (mean 60.34/67.48/71.15 um<sup>2</sup>, p=0.0011) and total actin signal (mean 38.15/46.01/45.49%, p=0.0255) compared to normoxia (Fig 1D-F). AAV neutrophils (n=8) were significantly larger, with more F-actin at baseline and showed a larger increase in actin following ANCA stimulation compared to HC cells: area (mean 75.73/98.68/121.25 um<sup>2</sup>, p=0.0088) and total actin signal (mean 51.74/74.85/83.44%, p=0.0032) (Fig 1G-I). <h3>Conclusion</h3> Neutrophils from patients with active AAV display distinct physical properties, which correlate with disease activity. Similar results are seen when neutrophils are stimulated <i>in vitro</i> with ANCA. In keeping with this, increased neutrophil actin polymerisation and marked lamellopodia formation is seen following ANCA stimulation <i>in vitro</i>, with the greatest effect seen when cells from patients with active AAV were used. Of interest, incubation under hypoxic conditions diminished the effect of ANCA stimulation suggesting that hypoxia can modulate actin polymerisation. This phenotype of increased cell stiffness in AAV may be due to reorganisation of the actin cytoskeleton and lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. Therefore, targeting cytoskeletal changes in AAV may offer a route to improve clinical outcomes. <h3>REFERENCES</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.